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Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data

ABSTRACT: We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced.

ORGANISM(S): Homo sapiens

PROVIDER: GSE21918 | GEO | 2010/05/21

SECONDARY ACCESSION(S): PRJNA129395

REPOSITORIES: GEO

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Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data

Project description:We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced. RBPs were UV-crosslinked to their RNA targets containing 4-thiouridine. The RNA segments were recovered after immunoprecipitation and seqeunced by Solexa.

2010-05-21 | E-GEOD-21918 | biostudies-arrayexpress

Advancing Functional Utility of PAR-CLIP by Quantifying Background Binding to mRNAs and lncRNAs

Project description:We developed a method for measuring non-specific background in PAR-CLIP data demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal. Furthermore, we show that quantitative determination of background is essential for identifying targets of weakly binding RNA-binding proteins and can substantially improve motif analysis. To define background binding events in PAR-CLIP data we performed the standard PAR-CLIP protocol (Hafner et al., Cell 2010.) on lysates expressing a commonly used non-RBP control, FLAG-GFP. After FLAG-tag immunopurification of UV 365nm irradiated lysates prepared from cells supplemented with 4-thiouridine (4SU), RNA was partially digested with RNase T1, radiolabeled and separated by SDS-PAGE. Reads were sequenced by Illumina HiSeq. PAR-CLIP was also performed for HuR. Included as well is a total from lysates treated like PAR-CLIP, but without immunoprecipitation (see sample description for more detail).

2013-11-07 | E-GEOD-50989 | biostudies-arrayexpress

Advancing Functional Utility of PAR-CLIP by Quantifying Background Binding to mRNAs and lncRNAs

2013-11-07 | GSE50989 | GEO

Confirmation of direct interaction between H3K56cr and GLYR1 by H3K*56cr Site-Link in living cells

Project description:As we have found that H3K56cr can crosslink with GLYR1 in living cells, we now perform H3K*cr Site-Link in GLYR1 overexpressed cells. The subsequent 365-nm light irradiation could activate the diazirine and covalently capture potential effector proteins on native chromatin in living cells. The crosslinked band could be identified after gel-based proteomics and pLink assisted MS/MS analysis of the crosslinked peptide.

2023-03-06 | PXD033960 | Pride

Photoactivatable-Ribonucleotide-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) of the RNA-binding protein (RBP) RBM4 in KBM-7 derived near-haploid human cell line, HAP1.

Project description:We hypothesized that RBM4 regulates HERVs by directly binding to their transcripts. To test this possibility, we performed photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). We performed four independent PAR-CLIP replicates of our own using HAP1 cells stably expressing a FLAG-tagged RBM4 (FLAG-RBM4) transgene under control of a doxycycline-inducible promoter. Following metabolic labeling with 4-thiouridine (4SU) and crosslinking with ultraviolet light (UV) of 312 nm wavelength, we isolated RNA covalently linked to FLAG-RBM4. The RNA recovered from four biological replicates was converted into cDNA libraries and deep sequenced.

2020-09-03 | GSE147895 | GEO

Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

Project description:The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.

2022-10-18 | MSV000090547 | MassIVE

eIF3 PAR-CLIP in 293T cells

Project description:Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis, and stress responses. The 13-subunit, 800-kDa eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photo-activatable crosslinking and immunoprecipitation (PAR-CLIP). eIF3 binds to a highly specific programme of messenger RNAs (mRNAs) involved in cell growth control processes, including cell cycling, differentiation, and apoptosis, via the mRNA 5' untranslated region (5' UTR). Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding cell proliferation regulators, c-Jun and BTG1, reveals that eIF3 employs different modes of RNA stem loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis. 293T cells were treated with 4-thiouridine and protein-RNA complexes were crosslinked, and eIF3-RNA complexes were immunoprecipitated.

2015-04-06 | E-GEOD-65004 | biostudies-arrayexpress

RNA-bound proteome of Mycobacterium bovis

Project description:The dataset accompanying a publication describing the RNA degradosome components in Mycobacterium tuberculosis (Mtb). The RNA-bound proteome was determined by affinity purification of protein-RNA complexes from Mycobacterium bovis cells fed with 4-thiouridine and crosslinked via photoactivation at 365nm. Affinity purified RNA-protein complexes were subjected to LC-MS analysis

2019-04-01 | PXD012661 | Pride

CLIP-Seq of human to identify the global RNA binding sites for TIA1 and TIAL1 proteins

Project description:Individual nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) and individual nucleotide resolution UV-crosslinking and affinity purification (iCLAP) were used to identify the global RNA binding sites for TIA1 and TIAL1 proteins. HeLa cells were UV crosslinked before lysing and digested with DNase and low concentration of RNase I. The protein-RNA complex were either immunoprecipitated with specific antibodies against either TIA1 or TIAL1 proteins and ligated to 3 prime adapter before separated by SDS-PAGE. For iCLAP, the His/Strep tagged proteins were overexpressed in HeLa cells, and the protein-RNA complexes were purified via Strep and His tag affinity purification. The proteins were digested by proteinase K and the RNA was reverse transcribed and self-circularised. The cDNA library was prepared by PCR with solexa primers compatible for high-throughput sequencing. This method allowed specific identify of crosslinked nucleotides, and genome-wide targets for TIA1 and TIAL1 not identified before. This method also allowed comparison between the 2 homologous proteins, whose binding sites and functions could be redundent. iCLAP provided an independent way to validate the binding sites identified by iCLIP, since no antibody was used in the iCLAP method.

2011-07-28 | E-MTAB-432 | biostudies-arrayexpress

Site-Link for identification of chromatinized proteome defined by site-specific histone acylations in living cells

Project description:By coupling the mass spectrometry, we developed the Site-Link strategy based on the GCE method for analyzing the interacting proteome of histone lysine acylations in living cells. As K* could mimic the natural lysine to be recognized by natural lysine synthase, histones bearing the site-specifically incorporated K*acyl, should also be also integrated into nucleosomes and further compacted into chromatin similar as Kacyl with cell passaging. The subsequent 365-nm light irradiation could activate the diazirine and covalently capture potential effector proteins on native chromatin in living cells. The effector proteins could be identified after gel-based proteomics and MS/MS analysis of the crosslinked histone-effector complexes.

2023-03-06 | PXD033959 | Pride

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